Industrial application of glucose isomerase
In industrial starch degradation enzymes play an important role. The enzyme .alpha.-amylase is used for liquefaction of starch into dextrins with a polymerization degree of about 7-10. Subsequently the enzyme .alpha.-amyloglucosidase is used for saccharification which results in a syrup containing 92-96% glucose. The reversible isomerization of glucose into fructose is catalyzed by the enzyme glucose (or xylose) isomerase. The correct nomenclature of this enzyme is D-xylose-ketolisomerase (EC 5.3.1.5) due to the enzyme's preference for xylose. However, because of the enzyme's major application in the conversion of glucose to fructose it is commonly called glucose isomerase. The equilibrium constant for this isomerization is close to unity so under optimal process conditions about 50% of the glucose is converted. The equilibrium mixture of glucose and fructose is known as high fructose syrup.
Fructose is much sweeter to the human taste than glucose or sucrose which makes it an economically competitive sugar substitute.
Many microorganisms which were found to produce glucose isomerase, have been applied industrially. A detailed review of the industrial use of glucose isomerases has been given by Wen-Pin Chen in Process Biochemistry, 15 June/July (1980) 30-41 and August/September (1980) 36-41.
The Wen-Pin Chen reference describes culture conditions for the microorganisms, as well as recovery and purification methods for the enzyme. In addition it also summarizes the properties of glucose isomerases such as the substrate specificity, temperature optima and pH optima, heat stability and metal ion requirement.
Glucose isomerase requires a bivalent cation such as Mg.sup.2+, CO.sup.2+, Mn.sup.2+ or a combination of these cations for its catalytic activity. Determination of 3D structures of different glucose isomerases has revealed the presence of two metal ions in the monomeric unit (Farber et al., Protein Eng. 1 (1987) 459-466; Rey et al., Proteins 4 (1987) 165-172; Henrick et al., Protein Eng. 1 (1987) 467-475).
Apart from a role in the catalytic mechanism, bivalent cations are also reported to increase the thermostability of some glucose isomerases (M. Callens et al. in Enzyme Microb. Technol. 1988 (10), 695-700). Furthermore, the catalytic activity of glucose isomerase is severely inhibited by Ag.sup.+, Hg.sup.2+, Cu.sup.2+, Zn.sup.2+ and Ca.sup.2+.
Glucose isomerases usually have their pH optimum between 7.0 and 9.0. There are several reasons why it would be beneficial to use glucose isomerase at a lower pH value. Three of these reasons;
a) stability of the sugar molecules, PA1 b) adaptation both to previous and/or later process steps and PA1 c) stability of the enzyme, will be further described below to illustrate this.
a) Under alkaline conditions and at elevated temperatures the formation of coloured by-products and the production of a non-metabolizable sugar (D-Psicose) are a problem. The desired working pH should be around 6.0. Around this pH degradation of glucose and fructose would be minimal.
b) A lowered pH optimum is also desirable for glucose isomerase when this enzyme is to be used in combination with other enzymes, or between other enzymatic steps, for example in the manufacturing of high fructose syrups. In this process one of the other enzymatic steps, the saccharification by .alpha.-glucoamylase is performed at pH 4.5.
c) Most of the known glucose isomerases are applied at pH 7.5. This pH value is a compromise between a higher initial activity at higher pH and a better stability of the immobilized enzyme at lower pH, resulting in an optimal productivity at the pH chosen (R. v. Tilburg, Thesis: "Engineering aspects of Biocatalysts in Industrial Starch Conversion Technology", Delftse Universitaire Pers, 1983). Application of glucose isomerase at a pH lower than 7.5 could benefit from the longer half-life and, combined with an improved higher specific activity, would consequently increase the productivity of the immobilized enzyme at that lower pH.
From the above it can be concluded that there is need for glucose isomerases with a higher activity at lower pH values under process conditions.
Many microorganisms were screened for a glucose isomerase with a lower pH optimum. Despite many efforts, this approach did not lead to novel commercial glucose isomerases.
In order to be able to change pH-activity profile of glucose isomerases towards lower pH by protein engineering it is important to recognize the underlying effects which give rise to the rapid decrease in catalytic performance at acidic pH.
The role of metal ions in enzymes
Two different functions for metal ions in enzymes can be envisaged.
First of all metal ions can have a structural role. This means that they are involved in maintaining the proper 3D-structure and, therefore, contribute to the (thermo)stability of the enzyme molecule. An example of such a structural and stabilizing role is Ca.sup.2+ in the subtilisin family of serine proteinases.
Secondly, metal ions can act as a cofactor in the catalytic mechanism. In this case the enzyme activity is strictly dependent upon the presence of the metal ion in the active site. The metal ion may for instance serve as a bridge between the enzyme and the substrate (e.g. Ca.sup.2+ in phospholipase binds the phosphate group of the substrate) or it may activate water to become a powerful nucleophilic hydroxyl ion (Zn.sup.2+ --OH.sup.-).
Examples are the Zn.sup.2+ -proteases such as thermolysin and carboxypeptidase, carbonic anhydrase (Zn.sup.2+), phospholipase-A.sub.2 (Ca.sup.2+) staphylococcal nuclease (Ca.sup.2+) and alkaline phosphatases (Mg.sup.2+, Ca.sup.2+). Examples of alpha/beta barrel enzymes which require cations to polarize a carboxyl or a carbonyl group in order to transfer hydrogen are glucose/xylose isomerase (Mg.sup.2+), ribulose-1,5-biphosphate carboxylase/oxygenase (RUBISCO) (Mg.sup.2+), enolase (Mg.sup.2+), yeast aldolase (Mg.sup.2+, K.sup.1+), mandolate racemase (Mg.sup.2+), muconate cycloisomerase (Mn.sup.2+). In the presence of metal chelating agents (such as EDTA), these enzymes loose their activity completely.
The binding of metal ions in a protein molecule usually involves coordination by 4 or 6 ligands. Depending on the type of metal ion, different ligands are found. For instance magnesium and calcium are usually liganded by oxygen atoms from either a carbonyl group of the protein main chain, a carbonyl group from a glutamine or asparagine side chain or the carboxylate from an aspartic- or glutamic acid side chain. Zinc and copper ions are usually liganded by nitrogen atoms from a histidine side chain or the sulfur atoms from cystein and methionine.
Factors determining the pH dependence of an enzyme
The activity of an enzyme is dependent on the pH value of the aqueous medium. This dependence is caused by the (de)protonation of ionizable groups in the active site of the enzyme on the one hand, and ionizable groups of the substrate, or product (if present) on the other hand. Ionizable groups in proteins involve the side chains of the basic amino acids lysine, arginine and histidine (carrying a positive charge in the protonated form), and the acidic amino acids aspartic acid, glutamic acid, cystein and tyrosine (all carrying a negative charge upon deprotonation). Furthermore, the amino group of the N-terminus and carboxyl group of the C-terminus carry a positive and negative charge respectively. The pK.sub.a -values of some amino acids are depicted in Table 1.
TABLE 1. ______________________________________ Ionizable groups of amino acids as occurring in proteins [Cantor and Schimmel, 1980, Biophysical chemistry, W. H. Freeman, San Fransisco] pK.sub.a ______________________________________ Positive charge (base) N-terminus 7.5-8.5 Lysine 10.5 Arginine 12.5 Histidine 6.0-7.0 Negative charge (acid) C-terminus 3.0-4.0 Aspartic acid 3.9 Glutamic acid 4.3 Cystein 8.3 Tyrosine 10.1 ______________________________________
It should be realised that these pK.sub.a -values are valid for model compounds and that great variations both within and between different proteins occur, due to the specific environment of the ionizable group. Electrostatic effects are known to play a fundamental role in enzyme function and structures (see J. A. Matthew et al, CRC Critical Reviews in Biochemistry, 18 (1985) 91-197). The presence of a positive charge near an ionizable group will lower its pK.sub.a while a negative charge will cause an increase in pK.sub.a. The magnitude of the effect decreases with the distance between the ionizable group and the charge. Moreover, the magnitude of this decrease is dependent upon the dielectric constant of the medium. Especially catalytic residues may reveal pK.sub.8 -values which deviate from these averages (see for instance Fersht, Enzyme structure and mechanism, 1985, W. H. Freeman, New York).
The pH-dependence of an enzyme catalyzed reaction can be dissected into the pH-dependence of the Michaelis constant K.sub.m and the pH-dependence of the turn-over rate constant k.sub.cat (equivalent to V.sub.max). These parameters represent the binding of the substrate in the ground-state and transition-state respectively. The pH-dependent (de)protonation of amino acid side chains which affect the binding of both substrate forms, or which are otherwise involved in the catalytic event (e.g. proton uptake and release as in general base catalysis), therefore, determine the pH-activity profile of an enzyme.
For instance the protonation of the histidine in the catalytic triad of serine proteases (both the trypsin- and subtilisin family) is responsible for the loss of activity at lower pH-values (&lt;7). In this case, the pK.sub.a of the enzyme activity is directly related to the pK.sub.a of this histidine residue.
As a second example, the two aspartic acid residues in aspartyl proteases, such as pepsin and chymosin, can be mentioned. These groups determine the pH optimum of these proteases. The typical structural arrangement of the aspartic acids causes them to have different pK.sub.a -values leading to the bell-shaped pH-activity profile.
It is known that altering the surface charge by extensive chemical modification can lead to significant changes in the pH dependence of catalysis. However in many cases this approach leads to inactivation and/or unwanted structural changes of the enzyme because these methods are rather unspecific. Selective chemical modification of lysines in cytochrome c was shown to have an effect on the redox-potential (D. C. Rees, J. Mol. Biol. 173, 323-326 (1980)). However, these results have been criticized because the bulky chemical reagent used for modification could perturb the structure of the protein.
Using the 3D-structure of a protein to anticipate the possibility of structural perturbation and site-directed mutagenesis, it is possible to modify the charge distribution in a protein in a very selective way.
Fersht and coworkers have shown that it is possible to manipulate the pH-activity profile of subtilisin by site-directed mutagenesis (Thomas et al, Nature, 318, 375-376 (1985); Russell et al, J. Mol. Biol., 193, 803-813 (1987); Russel and Fersht, Nature 328, 496-500 (1987)). Introduction of negatively charged groups at 10-15.ANG. from the active site at the protein surface raises the pK.sub.a value of the active site histidine. Conversely, making the surface more positively charged lowers the pK.sub.a of the acidic groups. Changing either Asp99 at 13 .ANG.ngstroms or Glu156 at 15 .ANG.ngstroms from the active site to a lysine lowers the pK.sub.a of the active site histidine by 0.6 pH units. Changing both residues simultaneously to give a double mutant with a change of four charge units, lowers the pK.sub.a by 1.0 pH unit. It appears that changes in Coulombic interactions can be cumulative.
Glucose isomerase mutants
WO 89/01520 (Cetus) lists a number of muteins of the xylose isomerase which may be obtained from Streptomyces rubiginosus and that may have an increased stability. The selection of possible sites that may be mutated is based on criteria differing from the ones used in the present invention. More than 300 mutants are listed but no data are presented concerning the characteristics and the alterations therein of the mutant enzyme molecules.
Methodologies for obtaining enzymes with improved properties
Enzymes with improved properties can be developed or found in several ways, for example by classical screening methods, by chemical modification of existing proteins, or by using modern genetic and protein engineering techniques.
Site-directed mutagenesis (SDM) is the most specific way of obtaining modified enzymes, enabling specific substitution of one or more amino acids by any other desired amino acid.